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IZiNCG - International Zinc Nutrition Consultative Group

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					           I ZiNCG                                                                                No. 02         2007


                         Technical Brief
               Assessing population zinc status with serum zinc concentration
    Biochemical indicators are an objective and quantitative                   weeks or months. However, other factors can independently
means of assessing the zinc status of a population. They are                   affect the serum zinc concentration. For example, infection
useful for identifying populations and specific subgroups that                 can lower serum zinc concentration, while muscle break down
are at elevated risk of zinc deficiency and therefore can be                   during weight loss can liberate zinc to the circulation and
used to target interventions to specific high-risk groups.                     increase serum zinc concentration.
     Serum or plasma zinc concentration1 is the best available
biomarker of risk of zinc deficiency in populations, for a                          For these reasons, serum zinc concentration may not be a
variety of reasons described below. WHO, UNICEF, IAEA,                         reliable indicator of an individual’s zinc status. Nevertheless,
and IZiNCG jointly recommend the use of serum zinc                             the distribution of serum zinc concentrations among a
concentration for assessment of population zinc status [1].                    representative sample of a population can be used to assess
                                                                               the risk of zinc deficiency in that population. In addition,
     For the correct use of serum zinc concentration as an                     because the serum zinc concentration rises consistently in
indicator of zinc status, there are several important technical                response to zinc supplementation, this indicator can be
issues that must be considered regarding sample collection,                    used as evidence of successful implementation of a zinc
laboratory analysis, and interpretation of the data.                           intervention program [2].

Why use serum zinc concentration as an indicator of
                                                                               Technical issues concerning the collection and
zinc status?
                                                                               analysis of specimens for serum zinc concentration
Serum zinc concentration has some important characteristics                         Blood specimens should be taken from the vein of a
that make it a good indicator of zinc status for populations:                  representative sample of people among populations or sub-
                                                                               groups of interest, as defined by age, geographic region,
 i. it reflects dietary zinc intake;                                           socio-economic status, or other descriptors. More information
ii. it responds consistently to zinc supplementation; and                      on sample selection and sample size issues is available in the
                                                                                 st
                                                                               1 IZiNCG technical document [3].
iii. reference data are available for most age and
     sex groups.                                                                     If serum zinc concentration is used to assess the impact
                                                                               of a zinc intervention strategy, such as supplementation,
To date, serum zinc is the only biochemical indicator of zinc                  fortification or dietary diversification, it is important to schedule
status known to meet these criteria.                                           the final blood collection before the end of the intervention
                                                                               occurs.
     Experimental studies of dietary zinc restriction in adult                      Zinc is present in the serum in very low concentrations,
volunteers have found that the serum zinc concentrations of                    so any contamination from exogenous sources of zinc can
previously well-nourished individuals decline within a few days                dramatically alter the test results. Therefore, samples must be
or weeks after their zinc intake is severely restricted. Some,                 collected and processed using zinc-free needles, syringes,
but not all, studies of moderately restricted zinc intakes have                centrifuge tubes, storage vials, and transfer pipettes, while
shown that serum zinc declines, although the response takes                    avoiding the destruction of red blood cells—hemolysis—and
longer and is less consistent. Research also shows that serum                  contamination of specimens with ambient zinc in air or water,
zinc concentration consistently rises when individuals consume                 or by contact with the analyst. A detailed description of
zinc supplements in addition to their usual diet, regardless of                appropriate blood collection techniques and suitable materials
their initial serum zinc concentration [2]. Thus, there is strong              that can be used for processing the specimens is provided in
                                                                                     st
evidence to indicate that, in general, serum zinc concentration                the 1 IZiNCG technical document [3].
reflects a person’s usual zinc intake during the previous few                       Ideally, specimens should be collected according to a


1
 Zinc concentration can be measured in either blood serum or plasma; for the sake of simplicity this document uses the term “serum zinc” or “serum zinc
concentration” to refer to both serum and plasma specimens.
strict protocol that controls the time of day and fasting status of   Table 1: Suggested lower cutoffs for serum zinc concentration (ug/dL)
the specimen donor. Because it may not always be possible                          by age group, sex, time of day and time since last meal
to collect specimens at the same time of day from all subjects,
the time of the blood drawing should be recorded, so the                                             Suggested lower cutoffs for
resulting values can be adjusted statistically as necessary.                                          serum zinc concentration
Because it is not always possible to ensure that all subjects                                                 ( g/dL)1
have either fasted or eaten within a defined time period (for
children), the time of the previous meal also should be noted.             Time of day                <10 years                ≥10 years
Once the blood is obtained, it should be stored in a cool box                  and
or in a refrigerator until centrifuged to separate the serum or
                                                                          fasting status               Males and        Non-pregnant
plasma from the blood cells. This will reduce the introduction                                                                            Males
                                                                                                        females           females
of possible artifact into the final results due to transfer of zinc
from the blood cells to the serum or plasma. Ideally, the serum
                                                                            Morning,
or plasma should be separated from the cells within 20 to 30                                         not available           70             74
minutes. Following centrifugation, the serum or plasma should               fasting2
then be transferred to a screw-top vial for storage, under
                                                                            Morning,
refrigeration (for up to several days) or frozen, until analysis.                                          65                66             70
                                                                            non-fasting
     Zinc concentration can be measured by a number
of different analytic instruments, such as flame atomic                      Afternoon,
                                                                                                           57                59             61
absorption spectrometry, graphite furnace atomic absorption                 non-fasting
spectrometry, inductively coupled plasma atomic emission              1
spectrometry, and neutron activation analysis. The                        For conversion to mol/L, divide by 6.54.
                                                                      2
measurement method depends on the local availability of                   Fasting is defined as no food or beverage consumption for at least 8 hours.
these instruments and the desired level of precision.

Interpreting the results: reference values and suggested                         Ideally, an acute-phase protein indicative of infection
                                                                           or tissue damage should be measured to help interpret the
cutoffs for adequate serum zinc concentration
                                                                           results. C-reactive protein (CRP) and a-1 acid glycoprotein
    Results of the analyses should be compared with the                    (AGP) are acute-phase proteins that can be used for this
appropriate reference data for age, sex, time of day, and                  purpose. If the concentration of the protein is greater than the
time since last meal to ensure accurate interpretation of the              normal threshold, it could indicate underlying inflammation,
data. Results should be reported as means, ranges, and                     which reduces serum zinc concentration. When elevated
percentages below the appropriate reference cutoff values for              acute-phase protein levels are found, the corresponding
the population as a whole and for selected sub-groups, as                  zinc values can be adjusted statistically or eliminated from
described below.                                                           the database, although this latter approach may introduce
    Reference values for serum zinc concentration are based                selection bias in the results.
on results obtained from a large sample of presumably well                      Population assessment should be repeated periodically
nourished Americans who participated in the NHANES II                      at the time of general nutritional status surveys to monitor
survey and were free from infection on the day of the blood                changes in the risk of zinc deficiency and response to
sample and not taking any medications that may have                        intervention programs.
affected their results [4]. Because serum zinc concentrations
vary by age group, sex, time of day of the blood collection                This technical brief was prepared by Dr. Kenneth H. Brown
and fasting status of the individual, lower limits of normal (i.e.,        and was reviewed by members of the IZiNCG Steering
the 2.5th percentile) are presented separately for each of these           Committee.
categories, as shown in Table 1.

     IZiNCG recommends that if more than 20% of the
population (or population sub-group) has a serum zinc
concentration below the relevant cutoff, the whole population
(or sub-group) should be considered to be at risk of zinc
deficiency [3].
    Steps in measuring serum zinc concentration to assess population zinc status

                 Identify appropriate population group, calculate required sample size, and
                               select a representative sample of the population




                                Obtain ethical approval and informed consent




Sample collection                                               Data collection
- Clean subject’s skin with alcohol at site of the              - Age
  antecubital vein
                                                                - Sex
- Restrict occlusion of subject’s arm with
                                                                - Time of day
  tourniquet for < 1 minute
                                                                - Time since last meal
- Draw blood using stainless steel needle, and
  collect into trace element-free evacuated                     - Presence of symptoms of infection
  blood collection tubes                                        - Other confounding factors
- Avoid zinc contamination (see table 2)




        Sample preparation
        - Place blood sample in refrigerator or on ice and allow to clot 20-30 min
                                                        0
        - Centrifuge blood sample at 2000-3000 × g for 1 minutes and separate serum or plasma
        - Discard any obviously hemolyzed samples




         Sample storage
        - Store serum or plasma samples frozen (or in refrigerator if they are to be analyzed within 1-2
          days)




        Sample analysis
        - Dilute sample for zinc 5 to 10-fold in solvents such as 6% aqueous butanol or 10% nitric acid
        - Read sample zinc concentration using an available instrument with appropriate standard
          dilutions, in-house quality controls, and Standard Reference Materials
        - Consider the measurement of an acute phase protein (CRP, AGP)




        Data analysis
        - Use appropriate cutoffs depending on characteristics of study population (see table 1)
        - Correct for time of day or time since last meal unless sample collection was standardized
        - Adjust serum zinc concentration statistically if acute phase protein is elevated
                                            Table 2:   Precautions to avoid zinc contamination

             - Disposable polyethylene gloves, free of talc or other coatings, worn by those handling blood
               samples;
             - Samples processed in laminar flow clean rooms, laminar flow hoods, or otherwise clean, dust
               and smoke-free laboratory;
             - Stainless steel needles;
             - Anti-coagulants (if separating plasma) that are pre-screened for zinc;
             - Trace element-free polyethylene evacuated tubes, stoppers, and serum separators; should be
               pre-screened for zinc prior to use;
             - Pre-screened, polyethylene processing and storage vials;
             - Except for pre-screened disposable equipment, all equipment used should be de-
               contaminated by washing procedures (soaked for 24 hours in ultrapure 10-20% HCl or
               HNO3 solution and rinsed 3-4 times in distilled, deionized water);
             - All materials and equipment stored covered or sealed to avoid dust.




References
1. Executive summary. Recommendations for indicators of population zinc status. Report of WHO / UNICEF / IAEA / IZiNCG Interagency
     Meeting on Zinc Status Indicators. Food Nutr Bull, 2007;28:S399-S400.
2. Hess SY, Peerson JM, King J, Brown KH. Use of serum zinc concentration as an indicator of population zinc status. Food Nutr Bull, 2007;28:
    S403-S429.
3. IZiNCG. Assessment of the risk of zinc deficiency in populations and options for its control. Food Nutr Bull, 2004;25:S94-S203.
4. Hotz C, Peerson JM, Brown KH. Suggested lower cutoffs of serum zinc concentrations for assessing zinc status: reanalysis of the second
    National Health and Nutrition Examination Survey data (1976-1980). Am J Clin Nutr 2003;78:756-64.




About IZiNCG
                     IZiNCG is the International Zinc Nutrition Consultative Group whose primary objectives are to promote and assist efforts
                     to reduce global zinc deficiency through interpretation of nutrition science, dissemination of information, and provision of
                     technical assistance to national governments and international agencies. IZiNCG focuses on identification, prevention and
                     treatment of zinc deficiency in the most vulnerable populations of low-income countries. The Steering Committee of IZiNCG
                     consists of 11 well-recognized international scientists with longstanding expertise in zinc nutrition and public health programs.

                      IZiNCG secretariat                c/o Program in International and Community Nutrition
                                                        University of California
                                                        One Shields Avenue Davis, CA 95616, USA
                                                        Tel: +1 (530) 752 0814                      Fax: +1 (530) 752 3406
                                                        E-mail: IZiNCG@ucdavis.edu                  www.izincg.org

                                                Produced with financial assistance from the Micronutrient Initiative (MI)
                                                             and the International Zinc Association (IZA).
                                                       For more information about MI visit www.micronutrient.org
                                                            For more information about IZA visit www.iza.com

				
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